Unit 4: Molecular Genetics
Content Outline: DNA Biotechnology (4.4) - Part 1
I.
Genetic Engineering (The field of science dealing with manipulating genomes.)
A. Recombinant DNA is the major focus of genetic engineering.
1. In this process, DNA from two different sources is joined into one molecule.
B. Biotechnology (This term refers to the use of computers and other devices to help in performing science.)
1. DNA gene cloning is an example.
a. This process involves the bacterial plasmids and another DNA source.
b. A plasmid is a small ring of DNA found in bacteria in addition to the main large circular
DNA strand found in the nucleoid region.
II.
Bacterial Cloning Process. This is used for inserting single genes into bacteria.
A. Step 1: Restriction Enzymes are used to cut a DNA plasmid and DNA from other donor source.
1. Restriction enzymes cut DNA at specific nucleotide sequences.
The specific DNA sequence is referred to as the Restriction Site.
2. Genes of interest (and antibiotic resistance genes) are cut into Restriction Fragments.
3. When the DNA is cut, “Sticky Ends”, or single-stranded DNA ends are created.
4. The same restriction enzyme must be used on both the plasmid and the DNA donor source.
a. Therefore, the “sticky ends” of the plasmid and the genes of interest will match and can be
joined.
B. Step 2: Create conditions for bacteria to take up recombined plasmids (bacteria are now transformed).
C. Step 5: Incubate transformed bacteria in the presence of antibiotic to select for only transformed cells.
D. Step 5: Transformed bacteria reproduce by binary fission to achieve a large working population.
E. Outcome: Transformed cells (that have antibiotic resistance) can express the gene of interest.
III.
Polymerase Chain Reaction (PCR) – This process requires no organism in the production of new DNA
molecules.
A. Kary Mullis won a Nobel Prize in 1993 for the development of this process.
1. The process is used to turn a single molecule of DNA into a large, workable sample of 100%
identical DNA molecules. This is widely used in criminal forensics (Murder cases).
2. Nobel Prize is the top award a scientist could receive for their research. It would be like an MVP
award in sports or an Oscar for actors or a Grammy for singers.
B. Process
1. The DNA sample is placed in a PCR Thermal Cycler machine.
a. The machine uses heat, DNA Primers, enzymes and a constant supply of nucleosides to
build new DNA molecules that are identical in nucleotide sequence to the original
molecule.
b. First step: Heat is used to separate the DNA double helix so that replication can occur.
c. Second step: The attachment of a DNA Primer to the template DNA strand will start
replication.
d. Third step: The DNA polymerase enzyme works 5’3’ attaching nucleosides to the
growing “new” side of the replicated DNA molecule.
e. Fourth step: Cool the mixture to recombine and stabilize the DNA back into a double
strand.
f. Repeat the cycle many more times to get large, workable sample of the DNA.
g. Perform a Gel Electrophoresis test to separate for comparison of nucleotide sequences
between organisms.
Part 2
I.
Gel Electrophoresis
A. This process is used to create a “DNA fingerprint”.
B. Different DNA samples are exposed to the same restriction enzyme.
1. This creates Restriction Fragment Length Polymorphisms (RFLP’s)
a. These are fragments of DNA having different lengths that were created using
restriction enzymes. (Can you see that in the term?)
C. The DNA RFLP’s are loaded into an agarose gel.
D. Turn on the electricity. (Remember, DNA is negatively charged because of the phosphate backbone, so it will
be repelled on the negative end [Black] and pulled by the positive end [Red].) (Electricity should flow from
the Black  Red strips when performing this process.)
E. The RFLP’s will separate according to length/size of the fragments.
1. Big pieces move slowly through the gel.
2. Small pieces move quickly through the gel.
F. The DNA fragments are stained for ease of viewing.
G. The DNA bands create a unique “fingerprint” of the individuals DNA.
II.
Human Genome Project (HGP)
A. The project was begun in 1990 and ended in 2003.
B. The project mapped out the entire DNA genome nucleotide sequence for all humans as a species.
C. The human genome contains approximately 40,000 different genes.
D. These 40,000 genes only make up about 3% of the total genome. That is amazing! Only 3%!
E. The other 97% are control sequences or introns(“spacers”).
III.
Transgenic Organisms
A. Recombined DNA from two different organisms are combined to make one organism that possess traits from
both “parent” organisms. These traits will be passed on through reproduction as the traits are in the DNA
nucleotide sequence, remember.
IV.
Applications (“uses”) of DNA Technology:
A. Gene Therapy
1. This uses a virus to introduce a new gene into a body cell’s DNA.
2. Somatic cells vs. Germ cells (Somatic cells only affect you; germ cells affect future generations.)
B. Pharmaceuticals
1. Helps with creating new medicines.
2. Vaccines against diseases and maybe even cancers in the future.
C. Criminal Forensics
1. DNA fingerprints of suspects.
2. Paternity/Maternity testing.
D. Environmental Clean-up
1. Bacteria are used to process human sewage in water treatment plants.
2. Bacteria that can clean up Oil Spills or breakdown Plastic by eating the oil compounds.
3. Organisms helping clean up heavy metals (such as Mercury) from mining or waste collection.
E. Agriculture
1. Engineering organisms to produce more and larger food.
2. Genetically engineering organisms able to produce hardier food for easy transport across the world.
3. Having organisms produce healthier foods.
4. Having organisms that can produce food during winter. (Winterized)
F. Livestock
1. Organisms that are “meatier”.
2. Organisms that are “leaner”. (Having less fat.)
3. Organisms that are disease resistant.